Papel de Las Fuerzas Sobre El Ligamento

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Periodontology 2000, Vol. 24, 2000, 5672 Copyright C Munksgaard 2000Printed in Denmark All rights reserved

PERIODONTOLOGY 2000ISSN 0906-6713

Role of physical forces inregulating the form and function of

the periodontal ligamentCHRISTOPHER A. G. MCCULLOCH, PREDRAG LEKIC & MARC D. MCKEE

The periodontal ligament is a physically small but

functionally important tissue in tooth support, pro-

prioception and regulation of alveolar bone volume.

There is a long-standing and widespread interest in

the periodontal ligament as a model connectivetissue because of its rapid matrix turnover and its

ability to adapt to alterations of mechanical loading.

These features are mediated in part by heterogen-

eous cell populations that enable the roots of teeth

to maintain dynamic yet strong attachments to bone

in spite of highly variable applied force levels. The

remarkably precise maintenance of periodontal liga-

ment width in spite of these force levels or the res-

toration of the ligament space after surgical ablation

indicates the existence of highly effective regulatory

systems for measuring tissue domains and forinitiating localized matrix resorption and synthesis.

Constitutive adaptation to applied forces is mediated

in part by specific structural and regulatory proteins

expressed by periodontal ligament cells. As an ex-

ample of these adaptations we review recent evi-

dence of how cytoskeletal proteins mediate protec-

tive responses to applied force and how these re-

sponses may enable the cells to survive in a

mechanically active environment.

Background

The rapid growth of interest in endosseous implants

as surrogates for natural teeth could indicate that the

demise of the periodontal ligament is imminent. As

many types of implants do not employ a gomphosis

to provide support and attachment to the jaw bone,

what is the rationale for continued study of the peri-

odontal ligament? We suggest that the many inter-

esting functional and biological features of this

tissue, its basic role in the development and main-

56

tenance of the periodontium and its core function

in the healing of periodontal wounds underlines its

fundamental importance. Further, the rapid re-

modeling of extracellular matrix proteins in the peri-

odontal ligament is the basis for its utility as a modelsystem to study connective tissue homeostasis and

remodeling. This chapter considers two discrete yet

interrelated aspects of periodontal ligament physi-

ology that underline its unique biological character-

istics and its central role in tooth support: 1) homeo-

static mechanisms for preservation of tissue do-

mains; and 2) adaptational features to high load

bearing. For definitive reviews on the general fea-

tures of the periodontal ligament, we refer you to

Schroeder (71), Berkovitz & Moxham (10) and Berko-

vitz (11). Asin vivo

data on the regulation of peri-odontal ligament function in humans are limited,

much of the work discussed in this chapter relates

to teeth of limited eruption in rodents or in non-

human primates and in vitro work on human peri-

odontal cells. While these models provide interesting

insights into the human periodontal ligament in

vivo, they are by no means perfect surrogates and,

consequently, it is not appropriate or desirable to ex-

trapolate directly from these data to the human situ-

ation.

Periodontal ligament structure andorganization

The periodontal ligament is a complex, vascular, and

highly cellular soft connective tissue that attaches

the tooth roots to the inner wall of the alveolar bone.

In general, all ligaments and tendons consist of par-

allel bundles of collagen fibers; some contain an ad-

ditional network of elastic fibers (such as ligamenta


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Periodontal ligament homeostasis

nuchae and flava) (86). The mechanical strength of

tendons and ligaments derives largely from the mol-

ecular structure of the type I collagen molecule and

its ordered arrangement into fibers (47). The fibers

of the periodontal ligament form a meshwork similar

to a stretched fishing net that extends between the

cementum and the bone. The fibers are anchored by

their insertion into bone or cementum as Sharpeysfibers. The mechanism by which collagen fibers are

tethered at their extremities is likewise important in

defining the overall mechanical properties of the

tissue system as a whole (16).

The periodontal ligament is unique among the

various ligament and tendon systems of the body in

that it is the only ligament to span two distinct hard

tissues namely, tooth cementum and bone (Fig. 1).

While tooth cementum has been likened to bone in

terms of function and morphology, it nevertheless

possesses unique anatomical and structural prop-

erties and is positioned at a soft tissuehard tissueinterface that is absolutely critical to the process of

mastication. Such an important anchoring tissue for

attachment of the periodontal ligament to the tooth

is mirrored on the wall of the alveolus in that bone

exhibits similar features in regard to structure and

composition; likewise, the periodontal ligament has

the ability to attach periodontal ligament fibers to

the alveolar process of the mandible and maxilla (15,

37, 43). Collectively, this arrangement forms a sus-

pensory complex involving two hard tissues and the

intervening soft connective tissue comprising the

periodontal ligament.

As might be expected for any biological system

routinely receiving repetitive biomechanical strains,

here associated with mastication, there are special

metabolic requirements and an architectural tissue

design that facilitate the function of the periodontal

ligament. Not surprisingly, such a unique and dy-

namic connective tissue system involving multiple

tissues requires exquisite regulation at the cellular

level. Maintenance and remodeling of periodontal

ligament collagen fibers (2123, 81), together with

the embedding and calcification of their extremitiesto form Sharpeys fibers (37, 43), requires the con-

certed action of numerous cell types (25) and

multiple, synchronized signaling mechanisms to co-

ordinate these activities. Central to these integrated

activities is the periodontal ligament fibroblast,

whose responsibilities include the formation and re-

modeling of the periodontal ligament fibers, and

presumably a signaling system to maintain peri-

odontal ligament width and thickness across the soft

tissue boundary defined by this ligament (53). Such

57

a central role in the physiology of the periodontium

dictates the need for precise cellular organization

and cellular signaling. In the periodontal ligament,

cellular signals are, in part, mediated by the forces

transmitted to the fibroblasts via collagen fibrils with

which they are in direct contact (25). Although not

particularly well characterized at the molecular level

specifically for periodontal ligament fibroblasts,some reports are available (33, 39, 78). Cell-matrix

interactions between fibroblasts and the extracellu-

lar matrix have been extensively studied and are re-

viewed elsewhere (69).

At the tissue level, periodontal ligament fibro-

blasts are rather regularly dispersed throughout the

ligament and are generally oriented with their long

axes parallel to the direction of the collagen fibrils

(Fig. 1). By virtue of their ability to both synthesize

and shape the proteins of the extracellular matrix,

periodontal ligament fibroblasts generate an organ-

izational tissue pattern in which collagen fibrils formbundles that insert into the bone and tooth ce-

mentum as Sharpeys fibers (16, 43, 56). This struc-

ture conforms to the three-dimensional architecture

of the periodontal ligament in a very precise manner

(20). At the level of the fibroblast cell body, the nu-

cleus occupies a large percentage of the volume of

the cell, but the surrounding cytoplasm contains the

full complement of organelles necessary to effect

protein secretion (7) (Fig. 2). During development

and the initial formation of the periodontal liga-

ment, the cytoplasm-to-nucleus ratio is high, and

fibroblasts appear very active in terms of having an

extensive network of rough endoplasmic reticulum,

a well-developed Golgi apparatus and abundant se-

cretory granules containing predominantly type I

collagen molecules destined for export. The cells

also develop long and thin cytoplasmic extensions

that form three-dimensional veils that compart-

mentalize the collagen fibrils into fibers. At all levels

of the cell, intimate connections are established be-

tween the plasma membrane and individual colla-

gen fibrils. Presumably, these sites represent contact

points for integrin-matrix linkages such that strainoccurring in the ligament is transmitted to the cell,

and appropriate cell signaling cascades are activated

(27). Typically, the collagen fibrils of the periodontal

ligament are quite uniform in size but show some

minor variability with age and at different anatomi-

cal locations within the periodontal ligament.

Several studies have indicated that the extracellu-

lar matrix collagens of the periodontal ligament have

an extremely high turnover and remodeling rate,

much higher than in gingiva, skin and bone (77).


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McCulloch et al.

Fig. 1. Light micrographs illustrating hard and soft tissue osteoblasts (Ob) at the crest of an inter-radicular alveolar

relationships in the periodontium. A. Low-magnification bone septum. D: dentin. DF. Age-related changes in peri-

image showing the histology of the periodontal ligament odontal ligament structure and organization. Early in its

(PL) and surrounding tissues. The periodontal ligament formation and just prior to tooth eruption (D), peri-

intervenes between the alveolar bone (AB) and the tooth odontal ligament formation by fibroblasts (F) forms co-

root, the latter consisting of dentin (D) and cementum. incident with extensive bone formation by osteoblastsOther nearby tissues include the pulp (P) of the tooth, (Ob) in each surrounding alveolus of the alveolar process

Hertwigs epithelial root sheath (HRS), marrow elements of the mandible and maxilla. Osteoblasts are positioned

(M) situated in the alveolar bone and the mandibular at the bone surface, whereas fibroblasts are dispersed

nerve (N). B, C. Higher magnification images showing throughout the developing periodontal ligament (PL).

periodontal ligament (PL) relationships with alveolar Collagen fibers are relatively small in diameter at this

bone (AB) and tooth. The collagen fibers of the peri- point, and their structure and insertions are not readily

odontal ligament span these two hard tissues, inserting visible. Capillaries (asterisks) are abundant at all stages of

into the matrices of bone on one side, and the cementum periodontal ligament development. At the time of tooth

(CEM) of the tooth on the other. Fibroblasts are abun- eruption (E), the periodontal ligament develops an obvi-

dantly dispersed throughout the periodontal ligament, as ous tissue organization such that collagen fibers are

are numerous capillaries. C illustrates bone formation by clearly defined and can be observed to insert as Sharpeys

58


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Turnover and remodeling in the periodontal liga-

ment imply synthesis and breakdown of matrix com-

ponents, particularly the collagenous fiber mesh-

work that extends between cementum and bone. As

indicated above, the collagen fibers of the peri-

odontal ligament form are critical for tooth support

and attachment to bone. They form a meshwork of

smaller fibers, each of which is composed of un-branched collagen fibrils that may run from one

fiber strand into another. It seems improbable that

one single fibril extends the entire dimensions of

tooth to bone, although there is no definitive evi-

dence for or against this view. Early work (72) sug-

gested that remodeling of the ligament is confined

largely to the mid-region of the periodontal ligament

where fibers from the bone and fibers from the tooth

interdigitate in an intermediate plexus. More re-

cent evidence suggests that this idea may not

necessarily be correct. Turnover and remodeling ac-

tivity in teeth of limited eruption, like the molars ofrodents, are found throughout the width of the peri-

odontal ligament from cementum to bone (6, 8, 66).

To adapt to changes of tooth position, the fiber sys-

tems in the periodontal ligament must be degraded

and new fibers synthesized. Since the periodontal

ligament is not made up of single strands of straight

collagen fibers but consists instead of a complex

meshwork, remodeling does not necessarily occur at

all sites synchronously. There is apparently some

fibers (arrows) into the alveolar bone (AB). Osteoblasts con-

form to this arrangement, and are clustered between the

insertion sites. Fibroblasts at this stage are abundant and

show a large amount of perinuclear cytoplasm housing the

extensive synthetic and secretory organelles expected for

such an active, collagen-producing cell. In association with

the organization of the periodontal ligament, bone re-

modeling occurs, as evidenced by the presence of cement

lines (CL) in the bone. In adult, mature periodontal liga-

ment of an erupted tooth (F), fibroblasts are more stellate

in appearance, and the volume of the periodontal ligament

that they occupy is less than at earlier stages, with extra-

cellular matrix (collagen fibers) being the predominant

component of the periodontal ligament. Although somesites in the alveolus show obvious insertions of Sharpeys

fibers (arrow) into bone, other surfaces of the alveolar bone

appear not to have obvious insertions of collagen fibers

into the bone. This observation may be related to the

timing of the bone remodeling cycle and the ability of resi-

dent fibroblasts and osteoblasts to act synergistically to

create locally new insertion sites. Nevertheless, a sufficient

number of biomechanically sound Sharpeys fibers exist at

any one time to accommodate the forces of mastication. D:

dentin. All samples are from post-natal rat periodontium

embedded in LR White and stained with toluidine blue.

Bars equal 50 mm.

59

flexibility in the system to permit adaptational

changes by breaking down short stretches of colla-

gen fiber bundles or single fibrils while leaving

others intact. This highly localized remodeling pro-

cess is undoubtedly facilitated by the phagocytosis

of collagen. Unlike the bulk removal of collagen that

is effected by extracellular matrix metalloprotein-

ases, collagen phagocytosis enables periodontal liga-ment fibroblasts to very precisely remove collagen

fibrils at specific sites (23).

While their roles in the periodontal ligament are

not yet clear, a number of reports have identified ad-

ditional extracellular matrix components including

collagen types V and VI, chondroitin sulfate, proteo-

glycans, fibronectin, tenascin and undulin (38, 48,

49, 91). An arborizing network of oxytalan fibers has

also been demonstrated in the periodontal ligament

and is most prominent in its occlusal half (7). In re-

lation to other ligaments and tendons, the peri-

odontal ligament is a highly vascularized tissue (13),and almost 10% of periodontal ligament volume in

rodent molar comprises blood vessels (53). This rela-

tively high blood vessel content may provide hydro-

dynamic damping to applied forces as well as pro-

vide high perfusion rates to this tissue.

Of particular importance to the function of the

periodontal ligament are its attachment points to

bone and tooth cementum (Fig. 3). At both sites, the

actual insertion of periodontal ligament fibers into

bone and cementum occurs as Sharpeys fibers, an

anatomical arrangement that represents the most

obvious means by which a tooth is retained in the

alveolus and in its occlusal plane. Once embedded in

either the wall of the alveolus or the tooth, Sharpeys

fibers calcify to a significant degree (36) and are as-

sociated with an abundance of noncollagenous pro-

teins commonly found in bone but also recently

identified in tooth cementum (17, 56). Notable

among these proteins are osteopontin and bone sial-

oprotein. Ultrastructural immunolabeling studies

using colloidal gold have demonstrated that high

levels of osteopontin accumulate at the insertion site

within the interfibrillar volume of the tissue (56), andit is thought that osteopontin and other proteins

contribute to the regulation of mineralization and to

tissue cohesion at these sites of elevated biomechan-

ical strain. Indeed, recent data directly demonstrate

that osteopontin is rapidly induced in alveolar bone

shortly after application of orthodontic forces to

teeth (84). A high concentration of noncollagenous

proteins relative to collagen could conceivably en-

dow unique and advantageous physical properties to

this critical hard tissuesoft tissue interface. Particu-


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McCulloch et al.

Fig. 2. Transmission electron micrographs and immuno- extensions (arrows) that envelop and define collagen fiber

cytochemical preparations illustrating the ultrastructure (CF) bundles. Nu, nucleus. B. Cross-sectional profiles of

of periodontal ligament fibroblasts, collagen fibrils, and the collagen fibrils (Coll) of mature periodontal ligament

cell-matrix relationships, and the intracellular pathway show them to be relatively uniform in diameter. C. Peri-

for collagen secretion. A. A transverse section through the odontal ligament fibroblasts show an extensive network of

periodontal ligament showing the stellate nature of peri- rough endoplasmic reticulum (rER) and a well-developed

odontal ligament fibroblasts (Fb) and their cytoplasmic Golgi apparatus with abundant stacked Golgi saccules

60


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larly for bone, remodeling activity successively

severs Sharpeys fibers to variable degrees as older

alveolar bone is replaced by new bone (37, 43). Con-

comitant with osteoclastic resorption, new connec-

tions are made in an asynchronous fashion such that

periodontal ligament fibers are continuously being

embedded in the alveolar wall.

Importantly, another architectural arrangement bywhich periodontal ligament fibers interface with the

bony surface has been identified. Here, Sharpeys

fibers are not readily identifiable as distinct bundles

within bone, but periodontal ligament fibers can be

observed to abruptly terminate at the bone surface

within a dark-staining band of variable, but generally

thin dimensions (37, 43). This band is rich in noncol-

lagenous proteins but is relatively poor in collagen

(Fig. 3). Based on its ultrastructure and protein com-

position as determined by immunogold cytochem-

istry, this band appears to represent a modified form

of cement line and is present at sites where collagenfibers of the periodontal ligament are directly ap-

posed to the bone surface and partly inserted into

this material. In all cases, this band of matrix is min-

eralized and rich in osteopontin, and may represent

a means by which a rapid connection of periodontal

ligament with the bone surface is established, al-

though it probably possesses less mechanical

strength. Full incorporation of the extremities of

periodontal ligament fibers at a later time would

likely require the concerted action of osteoblasts lin-

ing the alveolar wall.

(GS) showing typical, collagen-containing spherical dis-

tensions (SD) at their periphery. Transfer vesicles (TV) are

also common in this region of the cell. The plasma mem-

brane (PM) is in direct apposition with the collagen fibrils

(Coll) of the extracellular matrix. D, E. After post-embed-

ding, colloidal-gold immunocytochemistry on thin sec-

tions of periodontal ligament using antibodies raised

against mouse N-terminus collagen a1(I) to visualize in-

tracellular pathways for the production of type I collagenby periodontal ligament fibroblasts (Fb), gold particle im-

munolabeling indicates the presence of this protein in the

Golgi (G) region and secretory granules (SG) of these cells.

Ultimately, these a chains form the basis of the collagen

molecule, which assembles as collagen fibrils (Coll) in the

extracellular matrix of the periodontal ligament. rER:

rough endoplasmic reticulum; TV: transfer vesicles. Nu,

nucleus. Epon (AC) and LR White (D, E) sections of rat

periodontium stained with uranyl acetate and lead citrate.

Bars equal 5 mm (A) and 0.5 mm (B-E). Collagen antibody

courtesy of Paul Bornstein, Department of Biochemistry,

University of Washington, Seattle, USA.

61

Periodontal ligament cellpopulations

The healthy periodontal ligament contains several

discrete cell populations including fibroblasts, endo-

thelial cells, epithelial cell rests of Malassez, sensory

cells (such as Rufini-type end organ receptors),

osteogenic and osteoclastic cells and cementoblasts.The predominant cell type is the fibroblast, which

occupies about 30% of the volume of the periodontal

ligament space in rodents (8). The fibroblasts of the

periodontal ligament originate in part from the ecto-

mesenchyme of the investing layer of the dental pa-

pilla and from the dental follicle (82) and are differ-

ent from cells in other connective tissues in a num-

ber of respects. For example, the rapid degradation

of collagen by fibroblast phagocytosis is the basis for

the very fast turnover of collagen in the periodontal

ligament (23).

Although periodontal ligament cells are frequentlyconsidered as a homogeneous population, there are

some data indicating that the periodontal ligament

contains a variety of fibroblast populations with dif-

ferent functional characteristics (52). Whether these

subsets are derived from a single type of progenitor

cell is unknown. For example, the fibroblasts on the

bone side of the periodontal ligament exhibit more

abundant alkaline phosphatase activity than those

on the tooth side (32). Developmental differences

may also exist: Freeman & Ten Cate (24) and Ten

Cate (80) demonstrated that periodontal ligament

fibroblasts near the cementum are derived from the

ectomesenchymal cells of the investing layer of the

dental papilla, while fibroblasts near the alveolar

bone are derived from perivascular mesenchyme.

Cell kinetic experiments in rodent molar teeth

have shown that periodontal ligament cells comprise

a renewal system in steady state (51, 54). The rate of

cell renewal in the periodontal ligament is notable

for its rapidity and for the precise degree of regula-

tion in spatially discrete compartments. Periodontal

ligament progenitor cell populations undergo exten-

sive turnover in the maintenance of the steady stateand, in spite of extensive tooth drift or wounding,

many of these cells remain precisely located within

a narrow zone in the vicinity of small blood vessels

(30) and in the endosteal spaces of the adjacent al-

veolar bone (55). These cells proliferate, migrate and

ultimately produce more differentiated cells that can

synthesize bone, cementum and the extracellular

matrix of the periodontal ligament as has also been

shown in the developing periodontal ligament (62).

The generation of highly specialized cell popula-


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McCulloch et al.

Fig. 3. Immunocytochemical preparations selected to il- gen fibers (CF) insert as Sharpeys fibers (SF) into alveolar

lustrate the distribution of the noncollagenous protein os- bone, where they are tethered into a mineralized matrix

teopontin (OPN) at periodontal ligament collagen fiber in- rich in noncollagenous protein and containing abundant

sertion sites into alveolar bone and tooth cementum. osteopontin. Intense immunolabeling for osteopontin

A, B. The extremities of periodontal ligament (PL) colla- commences where the collagen fibrils insert (arrows) into

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tions that can remodel and heal damaged tissues in a

temporally and spatially appropriate manner is

thought to be essential for the repopulation and dif-

ferentiation responses in healing periodontium fol-

lowing extirpation of the periodontal ligament (58).

The signals that regulate these processes include cell-

matrix and direct cell-cell interactions which are

known to control cell proliferation, differentiationand cell function (34). For example, periodontal liga-

ment cells produce cell adhesion proteins like vi-

tronectin, tenascin and undulin as well as several

integrin subunits (78). Adhesive and cell-to-cell inter-

actions may be conducted through systemically

acting regulators that act on specific cell types that ex-

press the appropriate cognate receptor (83) or

through intercellular functions such as gap-junction-

mediated calcium fluxes (88). Indeed, the fibroblasts

of the periodontal ligament are connected by special-

ized junctional complexes which include as gap junc-

tions (7, 9, 76). While paracrine and autocrine regula-tion of periodontal ligament function is undoubtedly

important in mechanical stress-induced differen-

tiation of periodontal ligament fibroblast function

(50), recent evidence on mechanical stress-induced

DNA synthesis (41) suggests that autocrine function

may not beas important as other systems that may in-

clude electrical coupling. Thus the connectivity of

cells in the periodontal ligament may be of consider-

able importance in terms of the propagation of mech-

anically induced signals. Notably, mechanical stimu-

lation of the periodontal ligament stimulates the ex-

an otherwise mineralized bone matrix, which here has

been decalcified to highlight the underlying organic ma-

trix. At the insertion site, osteopontin appears to accumu-

late throughout the inter-fibrillar spaces and is likely in-

volved in regulating calcification at these sites and/or par-

ticipating in the maintenance of tissue cohesion. CL,

cement line. C. Where periodontal ligament (PL) collagen

fibers (CF) do not obviously insert for any distance into

the alveolar bone, they frequently abruptly terminate on

the bone surface in an osteopontin-rich, layer of organic

material (arrows) that resembles a cement line typicallyfound in bone. This arrangement may serve as an alterna-

tive attachment mechanism for the adhesion of collagen

fibers to bone in the absence of Sharpeys fibers. Fb,

fibroblast. D. On the tooth surface, Sharpeys fibers (SF)

exist where collagen fibers (CF) of the periodontal liga-

ment (PL) insert into cementum (CEM). Like for bone, os-

teopontin is a prominent constituent of these insertion

sites in cementum, with immunolabeling commencing at

the edge of the mineralized cementum (arrows) and con-

tinuing internally. Fb, fibroblast. LR White sections of rat

periodontium stained with uranyl acetate and lead citrate.

Bars equal 1 mm (A) and 0.5 mm (BD).

63

Fig. 4. Photomicrographs of rat periodontium including

the periodontal ligament showing the preservation of the

ligament width in young (A: 6 weeks) and old (B: 1 year)

rats. The sections were immunostained for bone sialop-

rotein (brown staining). AB: alveolar bone; PL: peri-

odontal ligament; C: cementum; P: pulp. Bar equals 100

mm.

pression of connexin 43, an important protein in the

formation of gap junctions in periodontal ligament

cells (79).

Regulation of periodontal ligamentwidth

The cells, vascular elements and extracellular matrix

proteins of the periodontal ligament function collec-

tively to enable mammalian teeth of limited erup-tion to adjust their position while remaining firmly

attached to the bony socket. Indeed some of the

most interesting features of the periodontal ligament

are its adaptability to rapidly changing applied force

levels and the capacity to maintain its width at con-

stant dimensions throughout the lifetime of the ani-

mal (53) (Fig. 4). This preservation of periodontal

ligament width throughout mammalian lifetime is

an important measure of periodontal ligament

homeostasis and gives insight into the function of


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McCulloch et al.

biological mechanisms that tightly regulate the met-

abolism and spatial locations of the cell populations

involved in the formation of bone, cementum and

the periodontal ligament. Cytokines and growth fac-

tors are important locally-acting regulators of cell

function and periodontal ligament cells are capable

of synthesizing and secreting some of these factors

(14, 18, 26). The ability of periodontal ligament cellsto synthesize and secrete a wide range of regulatory

molecules is an essential component of tissue re-

modeling and periodontal ligament homeostasis.

Notably, some of the transforming growth factor-b

isoforms synthesized by periodontal ligament cells

can induce mitogenic effects but can also dose-de-

pendently down-regulate osteoblastic differentiation

of periodontal ligament cells (18). On the other

hand, prostaglandins, which are also produced by

periodontal ligament cells, can inhibit mineralized

bone nodule formation and prevent mineralization

by periodontal ligament cells in vitro (60, 61). Peri-odontal ligament cells are also capable of regulating

bone formation by producing paracrine factors that

inhibit bone resorption (26). Conceivably, these mol-

ecules may modulate the osteogenic activity of peri-

odontal ligament cell populations and contribute to

the preservation of periodontal ligament width.

These types of cellular signaling systems may, there-

fore, be capable of accurately measuring and

maintaining the width of the periodontal ligament

in spite of high-amplitude physical forces during

mastication and despite the presence of osteogenic

cells within the whole width of the periodontal liga-

ment. Further, recent evidence has shown that the

pro-inflammatory cytokine interleukin-1 (75) and

one of the isoenzymes responsible for prostaglandin

synthesis (cyclooxygenase 2) (74) are induced by ap-

plied mechanical force on periodontal ligament cells

in vitro. As prostaglandins and interleukin-1 can

strongly induce matrix degradation, there is evi-

dently an important relationship between mechan-

ical forces, cytokine production and regulation of the

periodontal ligament space. The appropriate regula-

tion of these signaling systems is clinically importantsince the failure of homeostatic mechanisms to

regulate periodontal ligament width may lead to

tooth ankylosis and/or root resorption.

Experimental disruption ofperiodontal ligament homeostasis

Various experimental perturbations including des-

iccation (2), heat (46) and bisphosphonates (64, 87)

64

have been used to study homeostasis of periodontal

ligament width. These interventions rely in part on

the depletion of periodontal ligament cell popula-

tions or an apparent alteration of the differentiation

repertoire of periodontal ligament cells (44), disrup-

tion of periodontal ligament homeostasis and the

transient or permanent ingrowth of bone. Experi-

ments in dogs (40, 59), monkeys (1) and rodents (87)have shown that when periodontal ligament cells are

physically removed from the cementum or are per-

turbed by drugs such as the bisphosphonate 1-hy-

droxyethylidene-1,1-bisphosphonate, bone grows

into the periodontal ligament space and ankylosis

may occur. Although ankyloses can persist for long

periods of time, the tendency of the tooth root to

be resorbed and replaced by bone usually leads to

complete resorption over the long-term. As the peri-

odontal ligament is replaced with bone, propriocep-

tion is lost because pressure receptors in the peri-

odontal ligament are deleted or do not function cor-rectly. Further, the physiological drifting and

eruption of teeth can no longer occur and conse-

quently the ability of the teeth and periodontium to

adapt to altered force levels or directions of force is

greatly reduced.

To understand how periodontal ligament cell

populations restore their cellular and tissue do-

mains, appropriate model systems are required that

can provide insight into the origin of cells, their

regulation and differentiation. For this reason we

have used the rat periodontal window wound model

(Fig. 5) developed by Melcher (57) and modified by

Gould et al. (30). This model facilitates studies of

periodontal ligament homeostasis since precise por-

tions of the alveolar bone and the periodontal liga-

ment can be reproducibly deleted. Selective deletion

of the periodontal ligament and alveolar bone

causes a transient (60-day) disruption of the cellular

domains required to preserve homeostasis, thereby

providing a system to study the regulation of osteo-

genic cells by the adjacent periodontal ligament

cells.

To assess repopulation and differentiation of peri-odontal ligament cells in healing periodontal tissues,

we used combined 3H-thymidine labeling (31) and

immunostaining with a-smooth muscle actin, osteo-

pontin, alkaline phosphatase and bone sialoprotein

as differentiation markers of soft and mineralizing

connective tissue cell populations (45, 65). These

studies have shown that in contrast to ablation of

the periodontal ligament, preservation of the peri-

odontal ligament in the window wound model pro-

motes healing of the alveolar bone. Regardless of the


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type of wounding method a subset of periodontal

ligament cells express osteopontin, a widely recog-

nized but not wholly specific differentiation marker

of early bone formation. Immunostaining of peri-

odontal ligament cells for bone sialoprotein, a

marker of differentiated osteoblasts and cemento-

blasts, shows no staining reaction in periodontal

ligament cells under physiological or wounding con-ditions. The absence of this late marker of osteoblast

differentiation in repopulating periodontal ligament

demonstrates that, while a significant portion of

periodontal ligament cells may have osteogenic

characteristics (45, 67), these cells are blocked from

differentiating into osteoblasts. Consequently peri-

odontal ligament width is restored during the initial

healing phase (710 days, Fig. 6A) because osteo-

genic cells are unable to enter the mineralization

phase of osteogenic differentiation.

We have used bone morphogenetic protein-7 im-

plants in rat periodontal window wounds to probethe effect of this potent osteoinductive agent on

periodontal ligament cell differentiation (65). In

spite of the known ability of bone morphogenetic

proteins to induce ectopic bone formation in other

tissues (muscle) (85), periodontal ligament width is

preserved in healing periodontal tissues after

wounding. Bone morphogenetic protein-7 implants

are selective in promoting the proliferation and dif-

ferentiation of osteogenic cells but do not apparently

affect fibrogenic periodontal ligament cell popula-

tions, since the periodontal ligament width is un-

changed after administration of bone morphogen-

etic protein-7. However, administration of a bisphos-

phonate (1-hydroxyethylidene-1,1-bisphosphonate)

(44) reduces periodontal ligament width; this loss of

homeostasis could be the result of altered differen-

tiation of precursor cells in the periodontal ligament

and the recruitment of these cells into the osteo-

genic lineage. Notably, administration of 1-hydroxy-

ethylidene-1,1-bisphosphonate inhibits periodontal

ligament cell proliferative activity, reduces cell

counts and induces bone sialoprotein expression in

the body of the periodontal ligament, implying a dis-ruption of subsequent periodontal ligament cell dif-

ferentiation (Fig. 6B). Periodontal ligament width is

perturbed only after treatment with 1-hydroxyethyli-

dene-1,1-bisphosphonate for 2 weeks and occurs

after the reduction of periodontal ligament cell

counts, suggesting that a relatively small proportion

of non-osteogenic periodontal ligament cells is re-

quired for the maintenance of periodontal ligament

width.

To assess in more detail the requirement for speci-

65

Fig. 5. Diagrammatic representation of periodontal win-

dow wound through the buccal surface of the rat man-

dible. The wound model was originally developed by

Melcher (57) and modified later by Gould et al. (30). The

wound (W ) extirpates either alveolar bone alone or bone

and the periodontal ligament (PL). The regeneration of

the periodontal tissues following wounding provides an

excellent model to study the origin of the cells recoloniz-

ing extirpated periodontium and has been used exten-

sively for phenotyping of periodontal cells involved in re-

generation (44, 45) without microbial contamination from

the oral environment and without involvement of gingival

cells. AB: alveolar bone; C: cementum; P: pulp; D: dentine.

fic cell populations in tissue remodeling and the

preservation of periodontal ligament homeostasis,

we transplanted Lac-Z-positive murine periodontal

ligament cells into periodontal wounds of a Lac-Z-

negative animal (submitted manuscript). By trans-

planting previously characterized periodontal liga-

ment cells that express b-galactosidase (Lac-Z-posi-

tive cells) into periodontal wounds of rats not ex-

pressing this marker, we have examined the

differentiation in vivo of cells with a known initial


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McCulloch et al.

Fig. 6. A. Longitudinal section through rat periodontium ministered 1-hydroxyethylidene-1,1-bisphosphonate be-

including periodontal ligament (PL) and alveolar bone fore wounding. 1-hydroxyethylidene-1,1-bisphosphonate

(AB) stained for osteopontin. The periodontal ligament reduces cell counts in the body of the periodontal liga-

and bone were extirpated 10 days before sectioning. Note ment and causes the periodontal ligament width to

the staining for osteopontin in the newly formed bone shrink. Notably, periodontal ligament cells express bone

(NFB) and the restoration of normal periodontal ligament sialoprotein (blue staining in cells). The normal patterns

width. C: cementum. Bar equals 50 mm. B. In situ hybridi- of periodontal ligament homeostasis following wounding

zation for bone sialoprotein in a longitudinal section are lost, presumably because of switch of the periodontal

through rat periodontium as in A but animals were ad- ligament cell differentiation repertoire to an osteogenic

66


12/17


phenotype. Interestingly, at the first post-wounding

time (7 days), transplanted cells are present through-

out the entire periodontal ligament and at bone re-

modeling sites (Fig. 6C). Transplanted cells located

in the periodontal ligament exhibit the same pheno-

typic expression as had been determined in in vitro

cell cultures (osteopontin-positive, bone sialoprot-

einnegative). However, at day 14 and 28 afterwounding, transplanted periodontal ligament cells

are not found in the periodontal ligament but in-

stead are located at bone remodeling sites (day 14)

or in the outer layer of the regenerating bone (day

28, Fig. 6D, E). At these later time periods, the Lac-

Z-positive cells express bone sialoprotein, a marker

of a differentiated osteogenic cell. These data show

that transplanted Lac-Z-positive/bone sialoprotein

negative cells can, when embedded in the peri-

odontal ligament, differentiate into osteogenic cells

and migrate into appropriate bony sites. Evidently,

there are well-regulated systems that ensure cellswith the capacity to differentiate into osteoblasts are

restricted to existing bony sites. This regulation may

depend in part on physical loading of the peri-

odontal ligament, since unloaded teeth exhibit a

narrow periodontal ligament space.

Force distribution andmediators of periodontal ligament

remodelingPeriodontal ligament and alveolar bone cells are ex-

posed to physical forces in vivoin response to masti-

cation, parafunction, speech and orthodontic tooth

movement. Physiological loading of teeth or ortho-

dontically induced tooth movements involve re-

modeling of the periodontal and gingival connective

tissue matrices. Although the histological and some

of the biochemical effects of orthodontic force appli-

cation have been described, the mechanisms by

which applied forces produce reactive changes in

periodontal ligament and bone cells are poorly

phenotype. Bar equals 20 mm. CE. Transplanted Lac-Z-

positive periodontal ligament cells transplanted into

wounded rat periodontium. At 7 days after wounding (C),

transplanted cells marked with Lac-Z by histochemistry

are present throughout the periodontal ligament. At 14

days after wounding (D) or 28 days after wounding (E),

cells are associated with the margin of the alveolar bone

(AB) and begin to express bone sialoprotein. Bar equals

20 mm.

67

understood. Thus, while it is known that applied

mechanical force leads to more rapid bone remodel-

ing in vivo (68), knowledge of exactly how force dis-

tribution from the periodontal ligament to the al-

veolar bone regulates bone remodeling is meager. In

spite of these limitations, morphological obser-

vations of bone and periodontal ligament after ap-

plication of applied forces to mammalian teeth havelead to the following general suppositions: 1) the

periodontal ligament distributes applied forces to

the contiguous alveolar bone; 2) the direction, fre-

quency, duration and size of the forces determines

in part the extent and rapidity of bone remodeling;

3) when forces are applied to teeth devoid of a peri-

odontal ligament, the rate and extent of bone re-

modeling is very limited. These conclusions suggest

that the periodontal ligament may be both the me-

dium of force transfer and the means by which al-

veolar bone remodels in response to applied forces.

Progress over the last 10 years on force transduc-tion in biological systems has now been applied to

bone remodeling and to the role of the periodontal

ligament in force adaptation. Komatsu et al. (42)

have used a simple animal model system to examine

stress-strain functions in which root sections from a

variety of animal species are extruded from the al-

veolar bone. They found that the organization of

periodontal ligament collagen at particular sites in

the periodontal ligament is closely related to the

load characteristics in vitro. Andersen et al. (3)

examined stress and strain levels and their distri-

bution within the periodontium in a model system

based on human autopsy material. This model sys-

tem permitted an estimate of the stress levels that

may be distributed across the periodontal ligament

under applied loads. Mechanical forces can induce

fibronectin and collagen synthesis by periodontal

ligament cells in a strain magnitudedependent

fashion (35). These studies show in a reasonably di-

rect way that the metabolism and the organization

of the soft connective tissues of the periodontal liga-

ment are indeed modified by applied physical forces.

While applied loads may induce reactive changesin cells of the periodontium because of secondary

vascular and inflammatory effects, current evidence

suggests that periodontal ligament cells have a

mechanism to respond directly to mechanical forces

by activation of a wide variety of mechanosensory

signaling systems including adenylate cyclase,

stretch activated ion channels and by changes in

cytoskeletal organization. These alterations result in

the generation of intracellular second messengers

such as [Ca2]i, inositol 1,4,5-triphosphate and cyclic


13/17

McCulloch et al.

adenosine monophosphate. For example, [Ca2]i os-

cillations are generated in periodontal cells re-

sponding to substrate tension (4), and increased in-

ositol 1,4,5-triphosphate has been observed after

stretching of osteoblasts (19). Intermediate-term re-

sponses to applied force may include generation of

arachadonic acid metabolites. Indeed, the recent

demonstration of increased COX-2 expression bystretching of periodontal ligament cells in vitro (74)

suggests a mechanism by which increased prosta-

glandin levels may be generated. Interleukin-1 may

also be involved in periodontal ligament regulation

of bone remodeling in that cyclic-tension force

causes increased interleukin-1 production by human

periodontal ligament cells (75). Aging may exert a

modulating effect on interleukin-1 production since

in vitro aged periodontal cells produce more in-

terleukin-1 when stretched than younger cells (73).

Longer-term responses to mechanical loading in

vitro may include stimulation of cell division, al-though this response in periodontal ligament fibro-

blasts is apparently not due to an autocrine regula-

tory mechanism (41). Increased collagen synthesis

(35) and stimulation of alkaline phosphatase activity

(89) are also force-induced downstream changes that

likely impact on altering the form and function of

loaded periodontal ligament. These data indicate

that there are many potential routes by which ap-

plied loads to the periodontal ligament may lead

either directly or indirectly to alveolar bone remodel-

ing. Currently, much of the ongoing work on mech-

anotransduction has focused on signaling mechan-

isms, and there is great interest in determining the

nature of the mechanosensors in periodontal liga-

ment and bone cells.

Some of the most rapid responses in periodontal

fibroblasts subjected to mechanical strain in vitroin-

volve an elevation in intracellular calcium ions

([Ca2]i) (4), and changes in actin filament poly-

merization (63), which implies a fundamental role

for their modulation of subsequent intracellular

events. An increase in calcium-channel or nonspec-

ific cation-channel conductance would permit arapid elevation in [Ca2]i due to ion influx down a

strong electrochemical gradient. While earlier

studies investigating the mechanisms of physical

force transduction in periodontal tissues concen-

trated on the role of piezoelectric charges, the vas-

culature, cytokines and inflammatory mediators in

regulating the response of bone cells and fibroblasts

to mechanical forces, more recent studies have in-

vestigated the ability of these cell types to respond

directly to membrane perturbation. In periodontal

68

ligament fibroblasts, stretch of the cell membrane

induced by hypoosmolar cell volume increase can

activate stretch activated calcium permeable ion

channels, leading to an influx of calcium ions (12).

The influx of calcium ions can then strongly induce

other effectors including those proteins that regulate

the cytoskeleton.

The ability of actin filaments to rapidly reorganizein response to diverse external signals has been

demonstrated in cultured stromal cells in vitro. In

relation to physical stimuli, mechanical strain of at-

tached periodontal cells via a flexible substrate re-

duces filamentous actin content within 10 seconds,

which is followed by rapid polymerization (63). The

polymerization state of the sub-membrane cortical

actin meshwork may then affect the mobility and

function of cell surface receptors and could also me-

diate stretch-activated cation channel current (70).

Mechanoprotection

The actin-dependent sensory and response elements

of stromal cells that are involved in mechanical sig-

nal transduction are beginning to be clarified. To

study the role of actin in mechanotransduction we

have described a collagen-magnetic bead model in

which application of well-defined forces to integrins

induces an immediate (1 second) calcium influx

(28). We used this model to determine the role of

calcium ions and tyrosine-phosphorylation in the

regulation of force-mediated actin assembly and the

resulting change in membrane rigidity (27). Colla-

gen-beads were bound to periodontal cells through

the focal adhesion-associated proteins talin, vincul-

in, a2-integrin and b-actin, indicating that force ap-

plication was mediated through cytoskeletal ele-

ments. When force (2 N/m2) was applied to collagen

beads, confocal microscopy showed a marked verti-

cal extension of the cell, which was counteracted by

an actin-mediated retraction. Immunoblotting

showed that force application induced F-actin ac-

cumulation at the bead-membrane complex, butvinculin, talin and a2-integrin remained unchanged.

Atomic force microscopy showed that membrane

rigidity increased 6-fold in the vicinity of beads ex-

posed to force. Force also induced tyrosine phos-

phorylation of several cytoplasmic proteins, includ-

ing paxillin. The force-induced actin accumulation

was blocked in cells loaded with the intracellular cal-

cium chelator BAPTA/AM or in cells pre-incubated

with genistein, an inhibitor of tyrosine phosphoryla-

tion. Repeated force application progressively inhib-


14/17


ited the amplitude of force-induced calcium ion flux.

As force-induced actin reorganization was depend-

ent on calcium and tyrosine phosphorylation, and

as progressive increases of filamentous actin in the

submembrane cortex were correlated with increased

membrane rigidity and dampened calcium influx,

we suggest that cortical actin regulates stretch-acti-

vated cation permeable channel activity and pro-vides a desensitization mechanism for cells exposed

to repeated long-term mechanical stimuli. Thus, the

actin response may be cytoprotective since it

counteracts the initial force-mediated membrane ex-

tension and potentially strengthens cytoskeletal in-

tegrity at force-transfer points.

It seems self-evident that to survive in a mechan-

ically active environment, cells must adapt to vari-

ations of applied membrane tension. Part of this ad-

aptation involves sensing increases in extracellular

tension, maintaining contact with extracellular ma-

trix ligands and preventing irreversible membranedisruptions. Again with the use of the collagen-

coated magnetic bead model to apply forces directly

to the actin cytoskeleton through integrin receptors,

we investigated how the cytoskeleton reorganizes in

response to increased membrane tension. We found

that by a calcium-dependent mechanism, human

periodontal fibroblasts reinforce locally their con-

nection with extracellular adhesion sites (collagen-

coated beads) by recruiting actin binding protein-

280 into the cortical adhesion complexes (29). Actin

binding protein-280 was phosphorylated on serine

residues as a result of force application. This phos-

phorylation and the force-induced actin reorganiza-

tion were inhibited by bisindoylmaleimide, indi-

cating a role for protein kinase C isoforms. In a hu-

man myeloma cell line that does not express actin

binding protein-280, actin accumulation could not

be induced by force, while in stable transfectants ex-

pressing actin binding protein-280, force induced

actin accumulation similarly to human fibroblasts.

Cortical actin assembly evidently played an import-

ant role in regulating the activity of stretch-activated,

calcium permeable channels since sustained forceapplication desensitized these channels to sub-

sequent force applications and the decrease in

stretch sensitivity was reversed after treatment with

cytochalasin D. Further, in comparison to actin

binding protein-280-positive cells, actin binding

protein-280-deficient cells exhibited an almost two-

fold increase in stretch-activated channel activity

and significantly less (50% compared to 90% in actin

binding protein-280-positive cells) channel desensit-

ization following prolonged force application. Actin

69

binding protein-280-deficient cells showed a 90%

increase in cell death compared to actin binding

protein-280-positive cells (30% increase) after force

application, indicating a potential mechanoprotec-

tive role for force-induced actin binding protein-

280/actin reorganization. We suggest that actin bind-

ing protein-280 plays an important role in mechano-

protection by: 1) reinforcing the membrane cortexand thereby preventing force-induced membrane

disruption; 2) increasing the strength of cytoskeletal

links to the extracellular matrix; and 3) desensitizing

stretch activated ion channel activity.

Conclusions

Collectively, these data, which are largely from in vi-

tro investigations, indicate that stromal cells, and in

particular the fibroblasts and osteoblasts that popu-

late the periodontal ligament, have the necessarysignaling and effector mechanisms to both sense ap-

plied physical force and to mount a stream of re-

sponses which serve to maintain periodontal liga-

ment width and preserve cell viability. In the in-

stance of the periodontal ligament and the alveolar

bone these cellular characteristics have an important

consequence: the periodontal ligament is an abso-

lute requirement for rapid remodeling of alveolar

bone when physical forces are applied to teeth. This

requirement may be critically important for the

maintenance of alveolar bone volume following

tooth extraction since the loss of the periodontal

ligament terminates the mechanotransduction sig-

nals required for bone homeostasis.

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